Contraction of guinea‐pig isolated aorta induced by the prostaglandin E analogue sulprostone (1–400 nm) has a lower maximum response (40%) than that of phenylephrine or U‐46619 (TP‐receptor agonist). A prostanoid EP3‐receptor subtype is involved based on agonist potency ranking: equi‐effective molar ratios (EMR) are sulprostone (EC50∼23 nm) 1.0, SC‐46275 0.11, misoprostol 2.2, gemeprost 3.3, PGE2 5.4, 17‐phenyl PGE2 6.0, GR‐63799 8.9. GR‐63799, which contains a bulky ester group, is relatively more potent on neuronal EP3 preparations than on the aorta. ONO‐AP‐324, a relative of the non‐prostanoid prostacyclin mimetic series, behaves as an EP3 partial agonist on the aorta, inhibiting sulprostone responses but acting synergistically (in a similar manner to sulprostone) with phenylephrine; it may be a useful pharmacological tool for studying EP3‐receptors. Sulprostone contractions are markedly suppressed in zero‐Ca2+ bathing fluid containing either 2 mm EDTA or 50 μm EGTA, and by Cd2+ (500 μm), but are usually unaffected by nifedipine (0.3 μm) and verapamil (4.44 μm). Influx of Ca2+, but not through L‐type Ca2+‐channels, appears to be the major contractile mechanism. The guinea‐pig aorta is a valuable addition to the vascular EP3 preparations available and may increase our knowledge of the mechanisms whereby Gi‐coupled receptors mediate vasoconstriction (c.f. 5‐HT1B/D‐ and α2‐receptors). The possibility of certain EP3 agonists distinguishing EP3‐receptor isoforms is discussed. British Journal of Pharmacology (1998) 125, 1288–1296; doi:10.1038/sj.bjp.0702189